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Vitrification of multicellular systems and whole organs
580
ABSTRACTS, 24th ANNUAL
MEETING
94. The Mechanism of Action of Retrograde Oxygen Persufflation in Renal Preservation. D. E. PEGG, J. FOREMAN, C. J. HUNT AND M. P. DIAPER (MRC Medical Cryobiology Group, Department of Surgery, Douglas House, Tiumpington Road, Cambridge, United Kingdom).
nmol/mg protein), indicating that calcium accumulation did not cause the observed changes in mitochondrial function (Table 1). These results suggest that Ca plays a role in preservation damage and was tested in the transplant model. Kidneys preserved for 5 days with 0.5 mM Ca2+ and pretreated with a calmodulin inhibitor (chlorpromazine 2 mg/kg) had better function as determined by serum creatinines and survival (loo%, n = 5) than kidneys preserved with 1.5 mM Caz+ (survival 17%, n = 12) (Table 2). This study shows that the presence of too high or too low concentration of Ca2+ in the perfiisate is detrimental to mitochondrial function and that improved organ function and survival can be obtained by optimizing the perfusate calcium concentration and controlling intracellular calcium. Thus, calcium may be necessary to maintain structural integrity of the preserved organ but at high concentrations is damaging.
Retrograde oxygen persufflation (ROP) has been shown to improve the quality of preservation of kidneys that have suffered a period of warm ischaemia prior to storage. One might expect that oxygen would be acting through its role in oxidative phosphorylation, but in canine kidneys no increase in ATP or total adenine nucleotides was observed during storage. With rabbit kidneys a small but highly significant increase was observed, yet it was difftcult to understand how such a small amount of ATP could be important for subsequent function. We report studies with metabolic inhibitors in which we demonstrate that rabbit kidneys, at 0°C are able to trap chemical energy, and the ATP synthesized is utilized by the Na+/K+ pump, even at 0°C. There is, in consequence, less cell swelling in persufflated kidneys than in unpersufflated kidneys. These results demonstrate the continued turnover of ATP in rabbit kidneys at 0°C and the utilization of ATP to regulate cell volume, thus providing a rationale for ROP.
93. Hibernating Ground-squirrels as a Model for Cold Tolerance in Preserved Organs. C. .I. GREEN, B. J. FULLER*, S. SIMPKIN, AND B. LINSEY (Surgical Reserach Group, MRC Clinical Research Centre, Division of Comparative Medicine, Northwick Park Hospital, Harrow, Middlesex, HA1 3UJ and *Academic Department of Surgery, Royal Free Hospital, Hampstead, London). Kidneys were harvested from 13-lined groundsquirrels either while they were hibernating during the winter at an ambient temperature of 6-8°C or active during the summer months at an ambient temperature of 22°C. The organs were then flushed with hypertonic citrate solution and stored for various periods at 0°C. Renal slices assayed for in vitro gluconeogenic activity differed significantly: summer active kidneys exhibited zero activity after 5 days of storage while detectable levels of glucose production could still be found in hibernating kidney slices after 10 days of storage. The ultrastructure of the kidneys correlated well with physiological function. This degree of cold tolerance could not be matched in cold-acclimated rats. Kidneys from Colombian ground-squirrels, stored for 7 days at 0°C after vascular flush with hypertonic citrate solution, were autografted and were viable in 414nephrectomised recipients.
SYMPOSIUM VII-PROGRESS IN CELL, TISSUE, AND ORGAN PRESERVATION: BASIC AND APPLIED ASPECTS. PART II 95. Vitrijkation of Cell Systems. WILLIAM F. RALL (Research Laboratory, Rio Vista International, San Antonio, Texas). 96. Vitrification of Multicellular Systems and Whole Organs. GREGORY M. FAHY (Transplantation Laboratory, American Red Cross, Rockville, Maryland 20855). The ability to successfully cryopreserve organs would provide many advantages to the field of human organ transplantation. Vitrification is particularly attractive for organs due to their unusual susceptibility to injury consequent to ice formation. Many encour-
TABLE 2-Abstract 92 Effect of Ca2+ on Function of Kidneys Preserved for 5 Days (Serum Creatinines mg/dl f SEM) Davs uost TX
1
2
3
4
5
1.5 mMCa2+
3.7 f 0.2 2.5 f 0.3
5.8 k 0.2 2.8 ” 0.4
7.3 + 0.2 3.1 + 0.6
8.2 + 0.2 2.6 f 0.6
9.0 2 0.3 2.2 f 0.5
0.5 mMCa*+
7
6 1.9 f 0.4
1.8 k 0.5
8
9
10
-
-
-
1.5 f 0.3
1.3 2 0.2
1.2 + 0.2
1.1 2 0.2
ABSTRACTS, 24th ANNUAL aging reports of successful vitrification of simpler systems now exist, but serious attempts at the vitrification of organs have been prevented by limitations of fundamental knowledge and available technology. Recent developments, however, are about to change this situation. The construction of computer-directed organ perfusion equipment and associated software has recently made possible the addition and removal of vitrification solutions under well-controlled, repeatable conditions with continuous monitoring, display, and recording of appropriate data. The development of techniques for temporary transplantation of rabbit kidneys now allows realistic whole organ viability assessment during two hours of normothermic blood perfusion. Equipment required for high-pressure cooling and ultrarapid warming of human organs is also under construction. Recent results have indicated that many kidneys perfused with a new vitrification solution (VS4) under the correct conditions resume normal blood flows and substantial (69% of control) creatinine clearances (i.e., glomerular filtration rates, or GFRs) after a temporary initial period of vasospasm (“vascular crisis period”) possibly secondary to abnormal renin release. Absolute sodium and glucose reabsorptions are substantial (55% and 68% of control, respectively) and appear to be limited mostly by GFR. Leakage of protein to the urine currently averages about 4% of GFR at the end of the 120 min observation period. Potassium flux is qualitatively disturbed, possibly indicating leakage of intracellular K+ to the urine. For reasons which are not currently clear, 7 0u.t of 13 kidneys perfused with VS4 failed to resume function. These kidneys experienced a longer “vascular crisis period” than the kidneys that functioned, still having extremely depressed flows 30 min after transplantation. The release of renin from these kidneys was far more pronounced than the release observed for the functioning group and might explain the prolonged vascular crisis. Nevertheless, kidneys in this group ultimately resumed excellent renal blood flows. Histological findings in the functioning group were generally unremarkable in comparison to control histology, although material was more frequently found in some of the tubules in the VS4 group. Preliminary evidence suggests that most of the observed deficits in function seen in all VS4 kidneys is attributable to the temporary hypoxia caused by the vascular crisis, but hypoxia of this degree is known to be compatible with eventual life support function. One kidney has been cooled to below T8 after perfusion with VS4. Its near-equilibration with S4 was indicated by an almost complete absence of visible crystallization centers. These results suggest that renal perfusion and equilibration with a vitrification solution is not incompatible with life support function. If this conclusion can be confirmed by permanent transplantation studies, it may be possible to demonstrate the viability of cryopreserved rabbit kidneys in the near future.
MEETING
581
of Organ Cryopreservation. ARMAND KAROW (Department of Pharmacology, Medical College of Georgia, Augusta, Georgia).
97. Biophysics
SESSION VII-PROGRESS IN CELL, TISSUE, AND ORGAN PRESERVATION: BASIC AND APPLIED ASPECTS. PART II 98. The Mechanism of Damage during Hepatic Cryosurgery. B. RUBINSKY,* G. GNrK,t C. LEE,*
AND J. BASTACKY~ (*Department of Mechanical Engineering, University of California, Berkeley, California 94720; tAllegheny-Singer Research Institute, Pittsburgh, Pennsylvania 15212; SDonner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720). In the absence of an explanation for the mechanism of damage during freezing of tissue, it has been suggested that to ensure damage during cryosurgery the frozen tissue must be cooled to temperatures as low as - 50°C. Recently, hepatic cryosurgery which is the in situ destruction of liver tumors by freezing, has been made possible by the intraoperative ultrasound’s ability to visualize in real time the margin of the frozen lesion. Animal experiments and clinical treatment of tumors in the liver monitored by ultrasound suggest that tissue damage during hepatic cryosurgery corresponds much closer to the extent of the frozen region seen in the ultrasound than to the location of the - 50°C isotherm. In a companion paper it was shown that in the liver freezing occurs primarily through the vascular system and that water from surrounding cells will leave the cell through the membrane and also freeze in the vascular system. It was proposed that the consequent expansion of the vascular system and the dehydration of the cell are associated with damage to the tissue. This damage will increase at slower cooling rates. Studies were performed in which the temperature history during cryosurgery was evaluated analytically. The results show that during typical cryopreservation protocols the cooling rates at the edge of the frozen region are slow enough to ensure tissue destruction in that region if the cells become completely dehydrated. Since water transport through the cell membrane is a time dependent phenomena, to ensure maximal destruction of the tissue at the margins of the frozen region it is necessary to maintain the ice in the vascular system of that region long enough for complete cell dehydration. It should be emphasized that the mechanism of cell dehydration will occur in that region only if the water in the cells remains in a supercooled state and no intracellular nucleation occurs. Therefore, according to the studies on the mechanism of damage during freezing and on the thermal history during cryosurgery, to ensure tissue destruction it is not necessary to freeze to a very low temperature but rather to maintain the tissue that was frozen with slow cooling rates in a frozen state long enough for the
ABSTRACTS, 24th ANNUAL
MEETING
94. The Mechanism of Action of Retrograde Oxygen Persufflation in Renal Preservation. D. E. PEGG, J. FOREMAN, C. J. HUNT AND M. P. DIAPER (MRC Medical Cryobiology Group, Department of Surgery, Douglas House, Tiumpington Road, Cambridge, United Kingdom).
nmol/mg protein), indicating that calcium accumulation did not cause the observed changes in mitochondrial function (Table 1). These results suggest that Ca plays a role in preservation damage and was tested in the transplant model. Kidneys preserved for 5 days with 0.5 mM Ca2+ and pretreated with a calmodulin inhibitor (chlorpromazine 2 mg/kg) had better function as determined by serum creatinines and survival (loo%, n = 5) than kidneys preserved with 1.5 mM Caz+ (survival 17%, n = 12) (Table 2). This study shows that the presence of too high or too low concentration of Ca2+ in the perfiisate is detrimental to mitochondrial function and that improved organ function and survival can be obtained by optimizing the perfusate calcium concentration and controlling intracellular calcium. Thus, calcium may be necessary to maintain structural integrity of the preserved organ but at high concentrations is damaging.
Retrograde oxygen persufflation (ROP) has been shown to improve the quality of preservation of kidneys that have suffered a period of warm ischaemia prior to storage. One might expect that oxygen would be acting through its role in oxidative phosphorylation, but in canine kidneys no increase in ATP or total adenine nucleotides was observed during storage. With rabbit kidneys a small but highly significant increase was observed, yet it was difftcult to understand how such a small amount of ATP could be important for subsequent function. We report studies with metabolic inhibitors in which we demonstrate that rabbit kidneys, at 0°C are able to trap chemical energy, and the ATP synthesized is utilized by the Na+/K+ pump, even at 0°C. There is, in consequence, less cell swelling in persufflated kidneys than in unpersufflated kidneys. These results demonstrate the continued turnover of ATP in rabbit kidneys at 0°C and the utilization of ATP to regulate cell volume, thus providing a rationale for ROP.
93. Hibernating Ground-squirrels as a Model for Cold Tolerance in Preserved Organs. C. .I. GREEN, B. J. FULLER*, S. SIMPKIN, AND B. LINSEY (Surgical Reserach Group, MRC Clinical Research Centre, Division of Comparative Medicine, Northwick Park Hospital, Harrow, Middlesex, HA1 3UJ and *Academic Department of Surgery, Royal Free Hospital, Hampstead, London). Kidneys were harvested from 13-lined groundsquirrels either while they were hibernating during the winter at an ambient temperature of 6-8°C or active during the summer months at an ambient temperature of 22°C. The organs were then flushed with hypertonic citrate solution and stored for various periods at 0°C. Renal slices assayed for in vitro gluconeogenic activity differed significantly: summer active kidneys exhibited zero activity after 5 days of storage while detectable levels of glucose production could still be found in hibernating kidney slices after 10 days of storage. The ultrastructure of the kidneys correlated well with physiological function. This degree of cold tolerance could not be matched in cold-acclimated rats. Kidneys from Colombian ground-squirrels, stored for 7 days at 0°C after vascular flush with hypertonic citrate solution, were autografted and were viable in 414nephrectomised recipients.
SYMPOSIUM VII-PROGRESS IN CELL, TISSUE, AND ORGAN PRESERVATION: BASIC AND APPLIED ASPECTS. PART II 95. Vitrijkation of Cell Systems. WILLIAM F. RALL (Research Laboratory, Rio Vista International, San Antonio, Texas). 96. Vitrification of Multicellular Systems and Whole Organs. GREGORY M. FAHY (Transplantation Laboratory, American Red Cross, Rockville, Maryland 20855). The ability to successfully cryopreserve organs would provide many advantages to the field of human organ transplantation. Vitrification is particularly attractive for organs due to their unusual susceptibility to injury consequent to ice formation. Many encour-
TABLE 2-Abstract 92 Effect of Ca2+ on Function of Kidneys Preserved for 5 Days (Serum Creatinines mg/dl f SEM) Davs uost TX
1
2
3
4
5
1.5 mMCa2+
3.7 f 0.2 2.5 f 0.3
5.8 k 0.2 2.8 ” 0.4
7.3 + 0.2 3.1 + 0.6
8.2 + 0.2 2.6 f 0.6
9.0 2 0.3 2.2 f 0.5
0.5 mMCa*+
7
6 1.9 f 0.4
1.8 k 0.5
8
9
10
-
-
-
1.5 f 0.3
1.3 2 0.2
1.2 + 0.2
1.1 2 0.2
ABSTRACTS, 24th ANNUAL aging reports of successful vitrification of simpler systems now exist, but serious attempts at the vitrification of organs have been prevented by limitations of fundamental knowledge and available technology. Recent developments, however, are about to change this situation. The construction of computer-directed organ perfusion equipment and associated software has recently made possible the addition and removal of vitrification solutions under well-controlled, repeatable conditions with continuous monitoring, display, and recording of appropriate data. The development of techniques for temporary transplantation of rabbit kidneys now allows realistic whole organ viability assessment during two hours of normothermic blood perfusion. Equipment required for high-pressure cooling and ultrarapid warming of human organs is also under construction. Recent results have indicated that many kidneys perfused with a new vitrification solution (VS4) under the correct conditions resume normal blood flows and substantial (69% of control) creatinine clearances (i.e., glomerular filtration rates, or GFRs) after a temporary initial period of vasospasm (“vascular crisis period”) possibly secondary to abnormal renin release. Absolute sodium and glucose reabsorptions are substantial (55% and 68% of control, respectively) and appear to be limited mostly by GFR. Leakage of protein to the urine currently averages about 4% of GFR at the end of the 120 min observation period. Potassium flux is qualitatively disturbed, possibly indicating leakage of intracellular K+ to the urine. For reasons which are not currently clear, 7 0u.t of 13 kidneys perfused with VS4 failed to resume function. These kidneys experienced a longer “vascular crisis period” than the kidneys that functioned, still having extremely depressed flows 30 min after transplantation. The release of renin from these kidneys was far more pronounced than the release observed for the functioning group and might explain the prolonged vascular crisis. Nevertheless, kidneys in this group ultimately resumed excellent renal blood flows. Histological findings in the functioning group were generally unremarkable in comparison to control histology, although material was more frequently found in some of the tubules in the VS4 group. Preliminary evidence suggests that most of the observed deficits in function seen in all VS4 kidneys is attributable to the temporary hypoxia caused by the vascular crisis, but hypoxia of this degree is known to be compatible with eventual life support function. One kidney has been cooled to below T8 after perfusion with VS4. Its near-equilibration with S4 was indicated by an almost complete absence of visible crystallization centers. These results suggest that renal perfusion and equilibration with a vitrification solution is not incompatible with life support function. If this conclusion can be confirmed by permanent transplantation studies, it may be possible to demonstrate the viability of cryopreserved rabbit kidneys in the near future.
MEETING
581
of Organ Cryopreservation. ARMAND KAROW (Department of Pharmacology, Medical College of Georgia, Augusta, Georgia).
97. Biophysics
SESSION VII-PROGRESS IN CELL, TISSUE, AND ORGAN PRESERVATION: BASIC AND APPLIED ASPECTS. PART II 98. The Mechanism of Damage during Hepatic Cryosurgery. B. RUBINSKY,* G. GNrK,t C. LEE,*
AND J. BASTACKY~ (*Department of Mechanical Engineering, University of California, Berkeley, California 94720; tAllegheny-Singer Research Institute, Pittsburgh, Pennsylvania 15212; SDonner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720). In the absence of an explanation for the mechanism of damage during freezing of tissue, it has been suggested that to ensure damage during cryosurgery the frozen tissue must be cooled to temperatures as low as - 50°C. Recently, hepatic cryosurgery which is the in situ destruction of liver tumors by freezing, has been made possible by the intraoperative ultrasound’s ability to visualize in real time the margin of the frozen lesion. Animal experiments and clinical treatment of tumors in the liver monitored by ultrasound suggest that tissue damage during hepatic cryosurgery corresponds much closer to the extent of the frozen region seen in the ultrasound than to the location of the - 50°C isotherm. In a companion paper it was shown that in the liver freezing occurs primarily through the vascular system and that water from surrounding cells will leave the cell through the membrane and also freeze in the vascular system. It was proposed that the consequent expansion of the vascular system and the dehydration of the cell are associated with damage to the tissue. This damage will increase at slower cooling rates. Studies were performed in which the temperature history during cryosurgery was evaluated analytically. The results show that during typical cryopreservation protocols the cooling rates at the edge of the frozen region are slow enough to ensure tissue destruction in that region if the cells become completely dehydrated. Since water transport through the cell membrane is a time dependent phenomena, to ensure maximal destruction of the tissue at the margins of the frozen region it is necessary to maintain the ice in the vascular system of that region long enough for complete cell dehydration. It should be emphasized that the mechanism of cell dehydration will occur in that region only if the water in the cells remains in a supercooled state and no intracellular nucleation occurs. Therefore, according to the studies on the mechanism of damage during freezing and on the thermal history during cryosurgery, to ensure tissue destruction it is not necessary to freeze to a very low temperature but rather to maintain the tissue that was frozen with slow cooling rates in a frozen state long enough for the